Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Summary Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in the genes encoding the DNA repair proteins CSA or CSB. Csbm/m mice were given a high fat, caloric restricted or resveratrol supplemented diet. The high fat diet rescued the phenotype of Csbm/m mice at the metabolic, transcriptomic and behavioral levels. Additional analysis suggests that the premature aging seen in CS mice, nematodes and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high fat diet; and β-hydroxybutyrate, PARP inhibition, or NAD+ supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB is able to displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD+, through the deacetylase SIRT1 and suggests possible interventions for CS.

Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite.The Nudix hydrolases comprise a large family of proteins characterized by the highly conserved array of amino acids GX 5 EX 7 REUXEEXGU, where U represents a bulky, hydrophobic, amino acid, usually Ile, Leu, or Val (1). A recent BLAST (2) search of the sequence data banks has revealed more than 300 putative proteins from over 80 species containing this amino acid motif, the Nudix box ( Fig. 1). We have been systematically identifying and characterizing the enzymatic activities associated with these proteins, and we have found that almost all of the major substrates for these enzymes are nucleoside diphosphates linked to some other moiety, x, hence the acronym "Nudix." The range of substrates acted on by various members of the family includes ribo-and deoxyribonucleoside triphosphates, nucleotide sugars, dinucleoside polyphosphates, NADH, and ADP-ribose. These substances are potentially toxic to the cell, signaling molecules, or metabolic intermediates whose concentrations require modulation during changes in the cell cycle or during periods of stress. We have suggested that the role of the Nudix hydrolases is to sanitize or modulate the accumulation of these metabolites (1). Since the Nudix box is common to all of these enzymes, their specificity for the individual substrates must lie somewhere distal to the conserved region. In this paper, we describe the cloning and characterization of four ADP-ribose pyrophosphatases, and we identify a proline residue downstream of the conserved sequence common to members of this subfamily of Nudix hydrolases. Furthermore, we have observed that other recurring amino acids in this same region are predictive of two other subfamilies of the Nudix hydrolases, the dinucleoside polyphosphate pyrophosphatases and the NADH pyrophosphatases.We also demonstrate that ADP-ribose pyrophosphatase activity may play a role in tellurite resistance, since overexpression o...